Comparative Pharmacokinetics of Seven Major Compounds in Normal and Atherosclerosis Mice after Oral Administration of Simiao Yong'an Decoction

Simiao Yong'an decoction (SMYAD), a classic traditional Chinese medicine formula, has been used to treat atherosclerosis (AS) in clinical in China, but its therapeutic mechanism and pharmacodynamic material basis are not clear. In this study, the AS model was caused by a high-fat diet and perivascular carotid collar placement (PCCP), and SMYAD was orally administered to the model and normal mice. A rapid, sensitive, selective, and reliable method using ultrahigh-performance liquid chromatography (UHPLC) system combined with a Q Exactive HF-X mass spectrometer (UHPLC-Q Exactive HF-X MS) was established and validated for the simultaneous determination of seven compounds, including harpagide, chlorogenic acid, swertiamarin, sweroside, angoroside C, liquiritin, and isoliquiritigenin in the plasma of normal and AS mice. The specificity, linearity, precision, accuracy, recovery, and stability of the method were all within the acceptable criteria. The results showed that some pharmacokinetic behaviors of harpagide, chlorogenic acid, and isoliquiritigenin were significantly different among the two groups of mice. The specific parameter changes were harpagide (AUC0–t and AUC0–∞ were 11075.09 ± 2132.38 and 16221.95 ± 5622.42 ng·mL−1·h, respectively; CLz/F was 2.45 ± 0.87 L/h/mg), chlorogenic acid (t1/2 was 21.59 ± 9.16 h; AUC0–∞ was 2637.51 ± 322.54 ng·mL−1·h; CLz/F was 13.49 ± 1.81 L/h/mg) and isoliquiritigenin (AUC0–t and AUC0–∞ were 502.25 ± 165.65 and 653.68 ± 251.34 ng·mL−1·h, respectively; CLz/F was 62.16 ± 23.35 L/h/mg) were altered under the pathological status of AS. These differences might be partly ascribed to the changes in gastrointestinal microbiota, nonspecific drug transporters, and cytochrome P450 activity under the AS state, providing research ideas and experimental basis for pharmacological effects and pharmacodynamic material basis.


Introduction
Atherosclerosis (AS) is the significant pathological basis of cardiovascular and cerebrovascular diseases, which may cause stroke, coronary atherosclerotic heart disease, and myocardial infarction [1]. e pathogenesis of AS is complex and involves various factors, such as endothelial dysfunction, lipid deposition, smooth muscle cell proliferation, oxidative stress, cell apoptosis, and systemic and local inflammation [2].
According to the guideline, lipid-lowering drugs are the firstline drugs for the secondary prevention of coronary heart disease, and statins are the most widely used lipid-lowering drugs in clinical practice [3]. Statins have a significant effect on reducing blood lipid, but there are also adverse reactions, such as muscle symptoms and diabetes in long-term use [4]. Traditional Chinese medicine (TCM) contains a variety of active ingredients, which has good effects on the treatment and prevention of AS in clinical in China [5].
Simiao Yong'an decoction (SMYAD) is a traditional Chinese medicine formula, which consists of Lonicera japonica unb. (JYH), Scrophularia ningpoensis Hems. (XS), Angelica sinensis (Oliv.) Diels (DG), and Glycyrrhiza uralensis Fisch. (GC). SMYAD was used to treat gangrene in ancient times according to the traditional Chinese medicine classics. In recent years, this prescription has had a remarkable clinical effect in the treatment of atherosclerosis. It has been confirmed by pharmacological research that SMYAD can affect the migration of smooth muscle cells, inhibit the angiogenesis in plaques, and inhibit the release of inflammatory factors, antioxidative stress, but its pharmacodynamic material basis is indistinct [6,7]. Previous studies [8,9] have shown that SMYAD contains many kinds of compounds, such as iridoids, flavonoids, organic acids, and phenylpropanoids. Some of these components have anti-AS-related activities. Harpagide has an effect on facilitating cell migration into the inflamed tissue and promoting the anti-inflammatory activity of the resident macrophages [10]. Chlorogenic acid could protect the ApoE −/− mice against AS through accelerating the cholesterol efflux from macrophages [11]. Swertiamarin has been reported to have high antiatherogenic and cholesterol-lowering potential and can inhibit HMG-Co-A reductase [12]. Sweroside, angoroside C, and liquiritin can take many pharmacological roles, including anti-inflammatory, antioxidation, and cardiovascular protection [13][14][15]. Isoliquiritigenin could attenuate AS in ApoE −/− mice and inhibit the proliferation of human arterial smooth muscle cell [16,17]. Since plasma component concentrations and exposure times could well reflect the close relationship between the drug and its pharmacokinetic effect, it is important to interpret the blood components and pharmacokinetic changes for explaining prescription pharmacodynamic substances. Liu et al. investigated the pharmacokinetic characteristics following oral administration of extracts of SMYAD and its single and combined TCMs to rats [18]. However, the data on the pharmacokinetics of SMYAD are limited to normal physiological conditions, and its multicomponent pharmacokinetic investigation in disease conditions has been rarely conducted. e ultrahigh-performance liquid chromatography (UHPLC) system combined with a Q Exactive HF-X mass spectrometer can provide high resolution in a short analysis time, allowing the simultaneous detection and quantification of a large number of compounds [19]. erefore, we developed an analysis method for the simultaneous determination of harpagide, chlorogenic acid, swertiamarin, sweroside, angoroside C, liquiritin, and isoliquiritigenin in normal mice and ApoE −/− mice with a high-fat diet and perivascular carotid collar placement (PCCP)-induced AS using UHPLC-Q Exactive HF-X MS and conducted pharmacokinetic studies in normal and disease conditions. e results will discover the material basis of SMYAD for treating AS to support further drug development, and the pharmacokinetic differences between normal and model mice could provide a reference for the pharmacological mechanism. e AS model was established by a high-fat diet and perivascular carotid collar placement (PCCP) surgery in the ApoE −/− mice. After a 7-day adaptive feeding, the mice were given a high-fat diet for 2 weeks; before anesthesia, all ApoE −/− mice were fasted for 12 hours. During the surgery, the right common carotid artery was exposed, and a silicone cannula (length: 2.5 mm, inner diameter: 0.3 mm) was fixed around the carotid artery (external diameter: about 0.5 mm). Penicillin was injected intraperitoneally for 3 days after surgery to prevent infection.

Preparation of Stocks Calibration Standard and Quality
Control (QC) Samples. Stock solutions of linarin (internal standard, IS), harpagide, chlorogenic acid, swertiamarin, sweroside, angoroside C, liquiritin, and isoliquiritigenin were dissolved in methanol at the concentration of 100 μg/ mL, respectively. en the mixed stock solution was obtained by 6 μg/mL of harpagide, 8 μg/mL of chlorogenic acid, 48 μg/mL of swertiamarin, 10 μg/mL of sweroside, 2 μg/mL of angoroside C, liquiritin, and isoliquiritigenin. e IS working solution was diluted with the methanol to a final concentration of 400 ng/mL. Standard calibration curves were constructed by spiking 100 μL of blank mouse plasma with 25 μL of the standard working solutions and 25 μL of the IS working solution, yielding final plasma concentrations in the range of 2-1500 ng/mL for harpagide, 1-2000 ng/mL for chlorogenic acid, 5-12000 ng/mL for swertiamarin, 5-2500 ng/mL for sweroside, 5-500 ng/mL for angoroside C and liquiritin, and 2-500 ng/mL for isoliquiritigenin.

Preparation of Plasma Samples.
Each plasma sample (100 μL) was mixed with a threefold volume of methanol and 25 μL IS in a 1.5 mL EP tube. en the mixture was vortexed for 100 seconds and centrifuged at 12,000 rpm for 10 min at 4°C. e supernatant was transferred to another EP tube and evaporated to dryness under a nitrogen vacuum. e residue was reconstituted with 100 μL of 50% methanol and centrifuged at 15,000 rpm for 15 min. en the supernatant was transferred to sample vials for the LC-MS analysis.

Data Analysis.
e pharmacokinetic parameters, including maximum plasma concentration (C max ), time corresponding to C max (T max ), terminal elimination half-life (T 1/2 ), area under plasma concentration-time curve (AUC 0-t ) and area under the plasma concentration-time curve from 0 to infinity time (AUC 0-∞ ), apparent volume of distribution (Vz/F) and clearance (CLz/F), were calculated using the noncompartment model in DAS 2.0 software package (Shanghai, China). GraphPad prism 6.02 (Graph-Pad Software, USA) was used in the statistical analysis. All values are expressed as mean ± standard error. For the pharmacokinetic parameter values of the NC and AS groups, Student's t-test was employed for data comparisons. P values <0.05 were considered statistically significant.

ApoE −/− Mice as Model Were Successfully Established.
Serum biochemical parameters of mice including TC, TG, LDL-C, and HDL-C were determined (see Table 1). Compared to the normal group, TC and LDL-C levels were significantly increased in the AS model group (P < 0.01), and  Evidence-Based Complementary and Alternative Medicine there were no significant differences in TG and HDL-C levels between the two groups. HE staining (see Figure 2) showed that the NC group had intact carotid artery structure and no obvious AS plaque formation. Compared with the NC group, the AS group showed AS plaque protruding to the lumen, the vascular lumen was significantly narrowed, foam cells were piled up in the intima, the smooth muscle was disordered and migrated to the intima, and AS plaque was obviously formed. e above results indicated that the AS model was successfully established.

Specificity.
e selectivity was evaluated by comparing the typical chromatograms of blank plasma (in Figure 3(a)), the blank plasma spiked with the seven target analytes and IS (in Figure 3(b)), and the plasma after oral administration of SMYAD (in Figure 3(c)). ere was no endogenous interference in the full scan mode for each of the analytes in all samples, indicating the good specificity of the analysis method.

Linearity and Lower Limit of Quantification (LLOQ).
e calibration curves for the seven components were established by plotting the peak area ratios of each analyte to the IS against plasma concentrations using the least-square linear regression with a weighted (1/x) factor. e limit of detection (LOD) was determined for a signal-to-noise(S/N) ratio of more than 3. e lower limit of quantification (LLOQ) was determined as the lowest concentration on the calibration curve (S/N > 10). As shown in Table 2, the standard curves of the seven target analytes had good linearity with correlation coefficients more than 0.9969. e lowest LLOQ was 1.0 ng/mL.

Recovery and Matrix Effect.
e extraction recoveries and matrix effects of the seven compounds were evaluated by determining the QC samples at three concentration levels with five replicates. e recovery of six analytes was measured by comparing the peak areas obtained from the extracted QC samples with those obtained from mixed standards spiked into postextracted blank plasma. e matrix effect was expressed as comparing the peak response of the analytes in plasma samples with those of the pure standards prepared in methanol. e extraction recoveries were in the acceptable range from 90.17% to 111.33%. Regarding the matrix effects, all ratios were between 89.23% and 112.72%, suggesting that there were no endogenous substances and interference for the ionization of the analytes in the coeluting matrix (see Table 3).

Precision and Accuracy.
Precision and accuracy were evaluated as five replicate QC samples at LLOQ, low, middle, and high four concentration levels. Intraday precision and accuracy were analyzed during the same day while interday precision and accuracy were investigated on three successive days. Precision (relative standard deviation, RSD) and accuracy (RE) for intra-and interday values were below 15% and within ±15% for the seven compounds (see Table 4), respectively.
e results indicated that the intra-and interday precision (RSD%) of these analytes were in the range of 9.39% to 10.5%, respectively, while the corresponding REs ranged from −9.73% to 13.69%, respectively.
ese results suggested that this method had an acceptable precision and accuracy.

Stability.
Stability was evaluated as five replicate QC samples at two concentration levels (see Table 5). e extracted samples were stable under the testing conditions including autosampler temperature (4°C) for 24 h, room temperature (20°C) for 24 h, three freeze-thaw cycles, and long-term cold storage (−20°C for 30 days) with RE% values of −13.11% to 11.89%, indicating that all active ingredients were stable during the analysis.

Pharmacokinetic Study.
e validated method was successfully utilized to examine the concentrations of harpagide, chlorogenic acid, swertiamarin, sweroside, angoroside C, liquiritin, and isoliquiritigenin in NC and AS mice plasma. e mean blood concentration-time curves of seven active components after oral administration of SMYAD (35 g/kg/day) in NC and AS mice are illustrated in Figure 4, and the main pharmacokinetic parameters are listed in Table 6. e results suggested that active components harpagide, chlorogenic acid, swertiamarin, sweroside, angoroside C, liquiritin, and isoliquiritigenin were quickly absorbed in NC and AS mice after oral administration of SMYAD showing good pharmacokinetic parameters, which was consistent with previous reports [14,15,[20][21][22][23][24].
Compared with the normal mice, the trends of plasma concentration-time curves of the seven ingredients were changed in AS mice. In terms of pharmacokinetic parameters, the absorption of some active ingredients in AS mice was altered. Specifically, the AUC 0-t of harpagide (11075.09 ± 2132.38 ng·mL −1 ·h, P < 0.01), the AUC 0-∞ of hapagide (16221.95 ± 5622.42 ng·mL −1 ·h, P < 0.05), and the chlorogenic acid (2637.51 ± 322.54 ng·mL −1 ·h, P < 0.01) in   Evidence-Based Complementary and Alternative Medicine   Evidence-Based Complementary and Alternative Medicine  Evidence-Based Complementary and Alternative Medicine the AS group significantly increased compared with the normal group, indicating that harpagide and chlorogenic acid exhibited high absorption in AS status. For swertiamarin, sweroside, and angoroside C, the increased tendencies of AUC 0-t and AUC 0-∞ value were also observed, but no significant difference was discovered. However, significant reduction of AUC 0-t (502.25 ± 165.65 ng·mL −1 ·h, P < 0.01) and the AUC 0-∞ (653.68 ± 251.34 ng·mL −1 ·h, P < 0.05) for isoliquiritigenin in the AS group were discovered, indicating that the bioavailability of isoliquiritigenin was remarkably reduced in the AS group. For liquiritin, the decreased tendencies of AUC 0-t and AUC 0-∞ were also observed, but no significant difference was discovered. Furthermore, a longer T 1/2 for chlorogenic acid (21.59 ± 9.16 h, P < 0.05) wase observed in the AS group compared with the NC group, whereas no significant change in T 1/2 was observed for other compounds between the two groups. And the obvious lower CLz/F (P < 0.01) of harpagide and chlorogenic acid compared with NC group were observed, which means the metabolic rate of the two compounds slow down under pathological conditions. However, a significant increase in CLz/F (P < 0.05) of isoliquiritigenin was observed in the AS group. e results indicated that the oral administration of SMYAD could lead to slower elimination of chlorogenic acid and harpagide, but the elimination of isoglycyrrhizin was accelerated in AS mice. In addition, e Vz/F value of these compounds in both normal and model was large, indicating that the active compounds in SMYAD were possibly bound to tissue proteins, mainly distributed in tissue and intracellular fluids. e pathogenesis of AS is complex, and inflammatory reaction runs through the whole process of plaque rupture and thrombosis, especially unstable plaque rupture [25]. AS is often accompanied by a number of metabolic syndromes such as glycometabolism dysfunction and lipid-metabolism dysfunction. Under the pathological conditions of AS, the absorption, distribution, metabolism, and excretion of the main bioactive components of SMYAD may be affected by complex factors. e possible reasons for pharmacokinetic differences between normal and AS model mice may be addressed by the following explanations. It was reported that the abundance and composition of the gut microbiota were altered in AS patients [26].
e gut microbiota has the genetic mechanisms necessary to produce enzymes that metabolize oral drugs, focusing on two main types of reactions: hydrolysis and reduction. Microbial metabolism converts hydrophilic drugs into hydrophobic compounds, thereby enhancing their absorption in the gut, and the hydrolytic enzyme activities of intestinal flora may decrease in the atherosclerotic disease state [27], while the level of related proinflammatory factors (NF-κB, TNF-α, IL-1β, and IL-6) increases in AS [28]. Study [29] has shown that the increase of inflammatory factors will further reduce the expression of some enzymes, and these may be the cause of the slow metabolism of harpagide and chlorogenic acid, the increased absorption for harpagide and chlorogenic acid, and the decreased absorption for isoliquiritigenin in AS model mice. e cause of their absorption changes may also be relative to nonspecific drug transporters, such as P-glycoprotein. Changes in normal and AS status drug transporters can be further studied. Research [30] suggested that marked inhibition of hepatic cytochrome P450 activity in cholesterol-induced atherosclerosis in rabbits and decreased metabolism of harpagide and chlorogenic acid in AS mice may be related to the decrease in CYP450 activity in the disease state. All the above considerations suggested that the anti-AS mechanisms of SMYAD should be based on gut microbiota, drug transporters, and CYP450 activity.
Harpagide, chlorogenic acid, and isoliquiritigenin have anti-inflammatory, inhibition of oxidative stress, lowering blood lipids, and other pharmacological activities to ameliorate AS. erefore, it is speculated that these components may be the key active constituents of SMYAD in the treatment of AS. In addition, there was a large difference in mean blood concentration at some time points, and some pharmacokinetic parameters showed the trend of difference between normal and model, but it was not statistically significant, which may be the reason for the small sample size.

Conclusions
A sensitive, rapid, and reliable UHPLC-Q Exactive HF-X MS analytical method was established and validated for the simultaneous determination and quantification of harpagide, chlorogenic acid, swertiamarin, sweroside, angoroside C, liquiritin, and isoliquiritigenin in the plasma of normal and AS mice. It is the first comparative study of the pharmacokinetics of seven active components of SMYAD in normal and AS mice. e results demonstrated that AUC of harpagide and chlorogenic acid was significantly increased and CLz/F was decreased, meanwhile t 1/2 of chlorogenic acid was prolonged, while the AUC of isoliquiritigenin was decreased and its CLz/F was increased. Pharmacokinetic behaviors of swertiamarin, sweroside, angoroside C, and liquiritin were not changed obviously after oral administration of SMYAD in normal and AS model. is study laid the experimental foundation for further elucidating the pharmacodynamic substances of SMYAD against AS, and the difference in pharmacokinetic parameters provided a scientific reference for clarifying the targets of SMYAD in vivo.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare no conflicts of interest.

Authors' Contributions
Ke-han Sun and Man-fang Yang contributed equally to this work.